Extensive AIDS prevention programs and highly active anti-retroviral therapies have been successful in limiting the spread and the onset of the disease in high-income countries. However, it is generally agreed that global containment of the HIV/AIDS pandemics depends on our ability to develop prophylactic vaccines minimizing the viral transmission and infection. The long-term goal of this project is to develop an affordable, efficacious mucosal vaccine preventing HIV-1 transmission and early stages of infection. To this end, our efforts are focused on a novel HIV-1 virus-like particles (VLPs) consisting of the deconstructed HIV-1 Env protein (D-Env) and the Gag protein (D-Env/Gag VLPs). Towards the aim of economical production and mucosal delivery, we propose the expression of D-Env/Gag VLPs in plants. D-Env is composed of the external membrane proximal region residue 649-683 (MPR649-683), the transmembrane domain and the cytoplasmic tail of gp41. The specific hypothesis is that mucosally targeted D-Env/Gag VLPs can induce protective humoral and cellular immune responses at the mucosa, thereby synergistically blocking HIV-1 transmission and early stages of infection. We base that hypothesis on the findings by us and others that (i) antibodies targeting MPR649-683 were shown to block HIV-1 epithelial transcytosis and potentially neutralize the infection of CD4+ cells and (ii) Gag VLPs were shown to induce Gag-specific cytotoxic T cells in non-human primates. [unreadable] [unreadable] As an initial step to achieve the long-term goal, the specific aim of this proposal is to produce and in vitro characterize D-Env/Gag VLPs, which are designed based on the most pandemic HIV-1 subtype C, by the coexpression of D-Env and Gag in tobacco cells. In order to obtain high yield, the gag gene will be optimized for plant expression and the designed gene will be de novo synthesized. We will employ two expression systems, a modified tobacco mosaic virus (TMV)-based transient expression system and a stably transformed tobacco cell (NT1) suspension culture. These systems enable rapid investigations of the VLP assembly in plant cells. Upon successfully expressing Gag VLPs, efforts will be centered on creating the complete D-Env/Gag VLPs. This will be done by co-delivery of the plant optimized d-env gene with the gag gene in the TMV system and super-transformation of the Gag-expressing NT1 cells with D-Env expression vector. To characterize the plant-expressed D-Env/Gag VLPs, biochemical analyses by immunoprecipitation and pull-down assay, conformational analyses by light scattering and electron microscopy, and stability analysis using simulated gastric and intestinal fluids will be performed. The successful outcome of this project will not only provide a novel HIV-1 vaccine candidate that will be evaluated in the follow-up project using animal models, but also develop a new technology for its economical production. [unreadable] [unreadable] [unreadable]